Theriococcus barosii is a thermophilic organism obtained from deep vent flange, Endeavor Segment, Juan de Fuca Ridge, off the coast of Washington State in the U.S.A. Native Tba (nTba) DNA polymerase has been purified from cell paste by classical chromatographic purification methods including Q-Sepharose, Heparin Sepharose, phosphocellulose and Mono-Q (U.S. Pat. No. 5,602,011). Characterization of the purified nTba showed that it possesses an active proofreading function in addition to its DNA-dependent DNA polymerase activity. Tba DNA polymerase does not possess detectable 5'-3' exonuclease activity. Because nTba DNA polymerase was obtained from a thermophilic organism, it was shown to retain polymerase activity following prolonged treatment at elevated temperatures (95.degree. C.).
U.S. Pat. No. 5,602,011 discloses cloned Tba DNA polymerase.
Cloning of the gene for the Tba DNA polymerase DNA polymerase revealed that it is a member of the alpha-family of DNA polymerases and is approximately 80% conserved compared with the Pfu and Deep Vent DNA polymerases.
Based on amino acid sequence comparisons and protein expression studies on the truncated forms of other known polymerases, it can be shown that the organization of the gene is structured such that the 3'-5' exonuclease function is encoded in the 5' half of the gene, while the polymerase function is encoded in the latter half. The predicted amino acid sequence derived from the gene sequence highlights a metal binding site in the 3'-5' exonuclease domain. This domain is typified by the amino acid sequence FDIET and is conserved between Tba DNA polymerase, Pfu and Deep Vent. (See, for example, Vemori, et al., Nucl. Acids Research 21(2):259-265, 1993.)
Alteration of the FDIET sequence to FAIAT has been shown in some polymerases to eliminate the proofreading function. (Derbyshire, V., et al., Science 240:199-201, 1988; Bernad, A., et al., Cell 59:219-228, 1989; Frey, M. W., et al., Proc. Natl. Acad. Sci. USA 90:2579-2583, 1993; Mather, E. J., U.S. Pat. No. 5,489,523.)